Method and apparatus for testing microbial interaction with growth affecting substances

ABSTRACT

A method and apparatus for calculating the growth interacting substance potency and growth ratio for the intersections of a scanning spot with a track of visible microbial colonies on an interaction plate. The potency is a measure of the weight of a growth interacting substance which is deposited on the interaction plate per unit volume of the growth culture medium. The growth ratio is a measure of the growth interacting substance effect on growth of colonies of microbes on the interaction plate along the path of interaction of the scanning spot with visible microbial colonies.

CROSS REFERENCE TO RELATED APPLICATION

Reference is made to application Ser. No. 379,281 entitled Method forTesting Microbial Interaction with Growth Affecting Substances whichnames Samuel Schalkowsky and Ellen Schalkowsky as inventors and filed oneven data herewith.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to methods for measuring the effect of growthinteracting substances on microbial growth.

2. Description of the Prior Art

One well known test is the spot diffusion test in which a solution of agrowth interacting substance is applied to a culture medium which hasbeen inoculated with microbes. The resultant plate is incubated for aperiod of time sufficient to produce visible microbial colonies forareas of the plate where the potency of the growth interacting substancewas not high enough to inhibit growth. The spot test has knowndeficiencies. First, it does not provide direct quantitiveidentification of the potency of the growth interacting substance atpoints of interest on the incubated plate such as the point ofinhibition. Second it is limited by the degree of diffusibility ofgrowth interacting substances into the microbial culture medium.

A second well known testing method of growth interacting substancesinvolves the making of a series of solutions of increasing dilution ofthe growth interacting substance. Each solution is applied to amicrobial culturing medium which has been inoculated with a microbesolution and incubated for a time sufficient to detect the effect of thesolution on microbial growth. This method also suffers from knowndrawbacks. In the first place, it requires time consuming manualdilutions and requires the use of a large number of culture mediacontainers. It also does not readily yield precise quantitative data onthe potency of the growth interacting substance being studied. In thecase of its use to detect the point of inhibition, it usually revealsonly a range of potency between which growth inhibition occurs.

The spot and the dilution tests both suffer from the drawback of notreadily facilitating testing of growth interacting substances todetermine their effects in potencies which are less than those whichcompletely inhibit microbe growth.

The dilution method is also used to determine the effect of growthinteracting substances at less than inhibiting potencies by counting thenumber of surviving microbial colonies. The disadvantage of thisprocedure is that it requires separate tests since exposure times forsurvivor measurements are generally different than exposure times forinhibition measurement.

All of the above methods suffer from being time consuming because of theneed for manual manipulation and because they do not readily yieldcontinuous quantitive information on the potency of a growth interactingsubstance for diverse points of interest on a growth curve of themicrobes being studied.

Spiral Systems, Inc. manufactures an instrument under the trademarkSPIRAL PLATER for plating onto a microbial culture medium a solutioncontaining an unknown concentration of microbes to determine theconcentration of microbes in the solution by counting the resultantcolonies after an incubation period. The solution is deposited on acircular culture medium plate in an Archimedes spiral. The solutiondecreases in volume per unit length of the spiral as the radius of thespiral increases. The apparatus is described in U.S. Pat. Nos.3,799,844, 3,892,632, and 3,962,040. These patents are incorporatedherein by reference.

A laser bacteria colony counter (Model 500A) is manufactured by ExotechIncorporated, of Gaithersburg, Md., specifically for counting thecolonies which result from the plating of a microbe containing solutionwith the SPIRAL PLATER. The laser counter is the preferred microbecolony scanning mechanism which is used with the invention. The Model500A is described in a brochure entitled "User Manual Model 500A LaserBacteria Colony Counter, October 1981" by Spiral System Instruments,Inc. of Bethesda, Md., 20814. The description of the Model 500A isincorporated herein by reference.

The Food and Drug Administration pursuant to 21 CFR 036.105 requiresthat each manufacturer of antibiotics must run tests of each batch ofnewly manufactured antibiotic to determine compliance with potencystandards. Implementation of this requirement entails the measurement ofzones of inhibition of the test substance relative to the size ofinhibition zones produced by known potencies of control substances.Measurement of these zones of inhibition is done either manually or byvidicon scanners. These tests are costly in their need for extensivetime, materials, and equipment.

DEFINITIONS

1. Growth Interacting Substance--Any substance deposited in a programmedconcentration on a culture medium which either inhibits or inducesgrowth of microbes. Substances which inhibit growth of microbes arereferred to as growth inhibiting interacting substances and substanceswhich induce growth are referred to as growth enhancing interactingsubstances.

2. Control Plate--A plate containing a culture medium on which a trackof microbes has been deposited and incubated for a period of timesufficient to produce visible colonies along the track to provide areference track width for growth measurement in the absence of a growthinteracting substance.

3. Interaction Plate--A plate containing a culture medium on which aredeposited (1) microbes in the same track configuration and concentrationas the control plate, and (2) a growth interacting substance in contactwith the microbes. The plate is incubated for a period of timesufficient to produce visible colonies along at least part of the track.

4. Concentration--The number of microbes per unit volume contained in aliquid suspension which is deposited on the interaction and controlplates to produce the visible colonies of microbes.

5. Potency (Pi)--The weight of the growth interacting substance which isdeposited on the interaction plate per unit volume of the growth culturemedium.

6. Line of Intersection--The path followed by the spot of a scannerduring the intersection of the spot with a track of visible microbialcolonies on the interaction plate.

7. Length of Interception (Li)--The length that the spot of a scannerintercepts visible microbial colonies along the line of intersection ofthe spot with a track of visible microbial colonies. For continuousmicrobial colonies, the length of interception is the length of the lineof intersection of the spot with the track. For discontinuous microbecolonies, the length of interception is the sum of the separateintercepted line segements of the spot with discrete colonies along theline of intersection. The length of interception is a measure of thenumber of microbial colonies and their size. The size of a colony is ameasure of the number of bacteria in the colony and hence a measure ofbacterial growth rate.

8. Track Width (Wi)--The average width of a track of continuous (WC) ordiscontinuous (WD) microbial growth on the interaction plate along theline of intersection of the spot with the track. The width (Wi) isdefined by the equation Wi=Li×ΔR/2TiRi where Ri is the average radius ofthe spot measured from the center of the spiral, along the line ofintersection of the spot with the visible microbial colonies; ΔR is theradial distance between adjacent spirals of the microbial track, and Liis the length of interception. The discontinuous track width WD is lessthan the distance between the largest separation of visible colonies onopposite edges of the track of visible colonies.

9. Reference Width (Wo)--The width used in computing the growth ratioGRi. The reference width may be that measured from a control plate, itmay be the largest value of Wi measured on the interaction plate or itmay be an arbitrarily assigned value, e.g. Wo=1.

10. Growth Ratio (GRi)--The ratio of track width Wi and the referencewidth Wo.

11. Local Growth Measurement (LGM)--The measurement of track width Wimade during a single revolution of the scanner.

12. Regional Growth Measurement--The average value of a number ofcontiguous local growth measurements.

13. Growth Curve--The relationship of growth ratio, GRi, and Potency,Pi, for a particular combination of growth interacting substance andmicrobial population. A growth curve may be based either on local orregional growth measurement and is derived from those portions of theinteraction plate for which there is measurable microbial growth.

SUMMARY OF THE INVENTION

The invention is methods and an apparatus for determining the effects ofa growth interacting substance on microbial growth by utilizing aninteraction plate. The interaction plate contains visible microbialcolonies produced by the programmed deposition of a microbe containingsolution on a track on the plate, the programmed deposition of a growthinteracting substance on the interaction plate in contact with themicrobes and incubating the plate. In the first embodiment of theinvention, a point of interest along the microbial track on aninteraction plate, such as the point at which microbial growth is firstcompletely inhibited, is identified and the corresponding potency of thegrowth interacting substance is precisely determined from the knownprogram of the deposition of the growth interacting substance. Thesecond embodiment of the invention is based upon the discovery that thewidth of the track on the interaction plate of any point of interest maybe measured and compared with a reference width to produce a measurementof the effect of the growth interacting substance on microbial growth.The second embodiment of the invention preferrably uses control andinteraction plates in which each track is a spiral, the interactionplate has a gradient of deposition of the growth interacting substancewhich decreases in volume per unit length as the radius of the spiraltrack increases an the microbe containing solution concentration isconstant per unit length of the track on the control and interactionplates. The potency of the growth interacting substance at any point onthe track of visible microbial colonies may be calculated by use ofpublished figures of the known program deposition characteristics of theapparatus which applies the growth interacting substance to theinteraction plate. The growth ratio and potency of the growthinteracting substance may be analyzed for each line of intersection ofthe scanning spot with the track of visible microbial colonies.

A growth curve of the growth ratio versus potency of each growthinteracting substance may be made. The growth curve may be analyzed toquantitate selected growth curve information of interest such as slope,maximum, minimum, or intercepts with the GRi=0 axis, etc.

The first embodiment of the invention is a method for investigating theeffect of a growth interacting substance on microbial growth bydetermining the potency of the growth interacting substance at a pointof interest within visible colonies present on an incubated growthinteraction plate. The method includes the steps of applying a patternof a microbe containing solution in a pattern on an interaction plate;applying a solution of the growth interacting substance on theinteraction plate in a programmed potency in contact with the microbes;incubating the interaction plate for a time sufficient to producevisible microbial colonies on the interaction plate; and determining thepotency of the growth interacting substance at the point of interest bycorrelating the position of the point of interest within the visiblecolonies with the programmed volume per unit length of the growthinteracting substance deposited at that point. In the preferred form ofthe method, the patterns are Archimedes spirals; the potency of thegrowth interacting substance is a gradient which changes with the radiusof the pattern; and the concentration of the microbe containing solutionis constant per unit length of the spiral.

The method of the second embodiment of the invention includes the stepsof scanning an interaction plate with a scanner which scans a spot in aseries of adjacent paths to detect a length of interception of the spotwith visible colonies of microbes within a track along a line ofintersection of the spot with the track; calculating the average widthof the track of visible microbe colonies along the line of intersectionof the spot with the track from the detected length of interception ofcolonies which is a measure of the number of microbial colonies andtheir size; calculating the growth interacting substance potency at eachline of intersection of the spot with the track of visible colonies; andcalculating the growth ratio for each intersection of the spot with thetrack of visible colonies by calculating the ratio of track width forthe line of intersection of the spot with the interaction plate dividedby a reference width.

An apparatus in accordance with the second embodiment invention includesscanning means for scanning a spot in a series of adjacent lines fordetecting the visible colonies of microbes along a line of intersectionof the spot with the track of visible microbes; means responsive to thescanning means for detecting a length of interception of the spot withthe visible colonies of microbes along the line of intersection of thetrack of visible colonies which is a measure of the number of microbialcolonies and their size; means responsive to the scanning means forcalculating the average width of the track of visible microbial coloniesfor each line scanned by the scanning means; means for calculating thegrowth interacting substance potency at each line of intersection of thespot with the track of visible colonies; and means for calculating thegrowth ratio for each line of intersection of the spot with the track ofvisible colonies by computing the ratio of track width for the line ofintersection of the spot with the track of visible colonies and areference width.

The invention had advantages over the prior art methods and apparatus. Acontinuous measure of the interaction effects of growth interactingsubstances on microbial growth is produced. The continuous measure ofthe growth interaction effect enables identification of discrete pointsof interest anywhere on the growth curve of any growth interactingsubstance. Moreover, the precise detection of the point where microbialgrowth is inhibited by the growth inhibiting substance is facilitated.The study of the growth interaction effects on microbial growth isfacilitated at potencies where microbial growth is not totallyinhibited. The effects of a growth interacting substance on microbialgrowth may be studied over a wide range of potencies with a singleplate.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration of an interaction plate used with theinvention;

FIG. 1B is an illustration of a control plate used with the invention;

FIG. 2 is an illustration of an expanded section of FIG. 1A illustratingthe function of a scanner used with the invention;

FIG. 3 is a schematic of an apparatus used with the invention;

FIG. 4A is an illustration of a growth curve for a growth inhibitinginteracting substance obtained with the apparatus of FIG. 3;

FIG. 4B is an illustration of a growth curve for a growth enhancinginteracting substance obtained with the apparatus of FIG. 3; and

FIG. 4C is an illustration of a growth curve for a substance whichexhibits both growth inducing and growth inhibiting properties, obtainedwith the apparatus of FIG. 3.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1A illustrates an interaction plate 10 having visible microbialcolonies 12 in a growth pattern 14 in the form of a spiral. Theinteraction plate of FIG. 1A is representative of the interaction plateused when the invention is used to study growth inhibiting interactingsubstances in which the highest potency is applied near the center ofthe plate. It should be understood that the particular form of microbialgrowth pattern will vary with the gradient of the deposition of thegrowth interacting substance and the microbe containing solution andwhether the particular growth interacting substance is a growthinhibiting or a growth enhancing substance or a combination of both. Theinvention may be used with different forms of interaction plates otherthan the one used in FIG. 1A. The interaction plate 10 is made bypouring a solution of agar into a petri dish in accordance with knownmethods. The interaction plate 10 normally has the visible colonies in apartial spiral track 14. The plate has an inner circular area 16 inwhich no visible colonies appear because the growth interactingsubstance was applied with a potency which inhibited all growth. Theplate 10 also has an area of discontinuous microbial colony growth 18which includes an area 20 where all visible microbial colony growthapproaches zero (inhibition point), an area 22 where the visiblemicrobial colonies are discrete but of higher density, and an area 24where the number of colonies while still being discontinuous is denserthan area 22 with the greatest number of colonies appearing near theinside and outside boundaries of the spiral track 14 and the interior ofthe track having fewer visible colonies. The plate 10 also has an areaof continuous microbial colony growth 26 where the number of coloniesappears so dense that the colonies run together so that discretecolonies are no longer visible.

The interaction plate 10 of FIG. 1A is preferrably made by using acommercially available instrument manufactured by Spiral Systems, Inc.and marketed under the trademark SPIRAL PLATER to deposit an Archimedesspiral of a microbial solution in a constant concentration per unitlength of the track 14 and to deposit a solution of the growthinteracting substance in a programmed gradient which decreases withincreasing radius in contact with the microbes in the same Archimedesspiral as the microbial solution. The resultant interaction plate 10 isincubated for a period sufficient for visible microbial colonies toappear. The instrument is disclosed in U.S. Pat. Nos. 3,799,844,3,892,632, and 3,962,040. The instrument can be purchased with constantand variable cams which function as programs for respectively applyingthe test solution at a constant volume per unit length of the track 14and with a gradient which varies in volume per unit length of the track14. With the variable cam, the programmed gradient varies from thecenter of the plate 10 to its circumference in a ratio of about 40:1 forstandard 10 cm plates and 600:1 for standard 15 cm plates. The potencyof the growth interacting substance and concentration of microbes at anypoint on the track 14 may be calculated from the known programmedgradient of deposition and published information provided by SpiralSystem Instruments, Inc. One source of the published information is apaper entitled "Application of the Spiral Plating Method to BacterialInteraction Tests" by Samuel and Ellen Schalkowsky, which was presentedon May 27, 1981 at the 3rd International Symposium on Rapid Methods andAutomation In Microbiology and which is incorporated herein by referencein its entirety. Appendix A of the paper may be used to calculate thepotency of the growth interacting substance at any point on the spiraltrack 14 of a standard 10 cm plate. The information in theaforementioned appendix correlates the radius of the point on the spiraltrack 14 on which potency information is needed with the number ofmicroliters per millimeter squared deposited at that location by theSPIRAL PLATER. It should be understood that the invention is not limitedto the use of the SPIRAL PLATER and is not limited to the use of thecommercially available programs for producing potency gradients.

An important physical characteristic of the interaction plate 10 is thatthe width of the track 14 of visible microbial colonies decreases as thepotency of the growth interacting substance increases. In FIG. 1A, wherethe potency of the growth interacting substance was applied in a spiraltrack beginning near the center of the plate with an exponentiallydecreasing gradient, the width of the track 14 of visible microbialcolonies 12 increases inversely to the potency of the growth interactingsubstance applied to the spiral track. The invention measures the effectof the growth interacting substance on microbial growth by comparing theratio of the width of the track 14 at each point of interest with areference width Wo which may be the width of a track of a control plate.

FIG. 1B illustrates the control plate 28 which is used with theinteraction plate 10 of FIG. 1A to provide a reference of microbialgrowth when there is no growth interacting substance present. Thecontrol plate 28 is made by applying an Archimedes spiral of a microbecontaining solution in a constant concentration per unit lengthidentical to the solution applied to the interaction plate 10 of FIG.1A. The control plate 28 is incubated to produce continuous microbialcolonies 12 in an Archimedes spiral track 30. The width of the spiraltrack 30 is constant in view of the absence of a growth interactingsubstance. The width of the track 30 may be measured and stored inmemory as a reference width Wo as an input to the apparatus of FIG. 3.

FIG. 2 is an expanded view of a section of the interaction plate 10 ofFIG. 1A which illustrates the lines intersection 32, 34, and 36 of thescanning spot with the track 14. The inner radial end point of the linesof intersection 32, 34, and 36 is identified by the letter A and theouter radial end point is identified by the letter B. The lines ofintersection are the path of the scanner spot (FIG. 3) across the tracks14 and are of constant radius when a circular spot scanner is used or aslowly varying radius when a spiral spot scanner is used. Either type ofscanning path may be used with the apparatus of FIG. 3 with the spiralspot scanner being preferred in view of its commercial availability.When either a circular or a spiral scanner is used, the radius of thescanning spot decreases by a distance Δr for each successive revolutionof the spot scanner which causes the spot to start at the outside of theplate and scan toward the center. The zone of inhibition 16 is definedby a radius of substantially constant radius, such as radius R1, (lineof intersection 36). The reason that the zone of inhibition 16 issubstantially circular is that the interacting substance tends todiffuse from the point of application such that the resultant diffusionpotency gradient decreases with increasing radius thus causing the locusof points of equal potency to be defined approximately by a circle. FIG.2 illustrates the increase in widths with increasing radius of the track14 where W3>W2>W1 as stated above with reference to FIG. 1.

FIG. 2 illustrates parts of the incubated interaction plate 10 which areimportant in understanding the present invention. The inhibition tail 23is the area between points A and B respectively of the innermost visiblespiral of deposition. Several measured parameters of the incubatedinteraction plate of FIG. 2 are used by the apparatus of FIG. 3. Thequantity RS is the radius of starting point of the deposition of thegrowth interacting substance and the microbial-containing solution onthe interaction plate. The quantity RA is the distance from the startingradius RS to the point of interest illustrated as the inhibition point Bwhere microbial growth ceases of the innermost visible spiral ofdeposition. The radius of the spot scanner at any point of interest isequal to the sum of RS+RA. The radii R₁, R₂ and R₃ represent differentaverage scan radii of the spot scanner.

FIG. 3 illustrates the preferred apparatus of the second embodiment ofthe invention. A spiral spot scanner 40 is used to scan the interactionplate 10 of FIG. 1A described above. The spiral spot scanner preferrablyscans a laser beam in a spiral of decreasing radius over the interactionplate 10 to detect the visible microbial colonies 12 in the track 14.Although a circular spot scanner may be used with the apparatus of FIG.3, the preferred form of the scanner is a Model 500A spiral spot scannermanufactured by Exotech Incorporated of Gaithersburg, Md., which isreferred to in the Description of the Prior Art. The Model 500A hasoutputs 42, 44, and 46 which, respectively, are signals of eachintercept of the spot scanner with each microbial colony 12 (eithercontinuous or discontinuous), the internal clock signal of the scanner,which has 1024 clock pulses per scan revolution, and a signal indicatingthe detection of previously undetected microbial colonies 12. Theintercept output signal on line 42 is connected to an And gate 48. Theclock output line 44 is also coupled to the And gate 48. The totalnumber of outputs of the And gate 48 per scan revolution is a functionof the length of the line of intersection of the spot during which thespot is intercepting visible microbial colonies. When continuousmicrobial colonies 12 are being intercepted, such as when scanning alongline of intersection 32 of FIG. 2, the length of interception is equalto the length of the line of interception AB. When discontinuous microbecolonies 12 are being intercepted, such as with the scanning along lineof intersection 36 of FIG. 2, the length of interception is less thanthe length of the line of interception AB and is proportional to thedensity of the microbial colonies along the line of intersection as willbe described infra. The length of the line of interception is used tocalculate the average width Wi of the track 14 along the line ofintersection of the scanning spot. The intercept output 42 is alsocoupled to intercept counter 50 which functions to count the totalnumber of intercepts during each revolution of the scanning spot ofscanner 42. The scanner clock 44 output is also coupled to a scanrevolution counter 52 which contains an internal counter which providesan end of scan revolution signal on line 54 when the count total equalsthe number of clock pulses N per revolution of the scanner 40. When theExotech Model 500A laser spot scanner is used, the scan revolutioninternal counter counts to 1024 to signal the completion of a scanrevolution. The end of scan revolution signal of line 54 is applied tothe intercept counter 50 which has been previously described tosynchronize the counting of intercepts with the completion of a scanrevolution. The output from the intercept counter 50 on line 56 isconnected to the output computation block 58. The total count stored inthe intercept counter 50 is monitored by the output computation block 58to detect when the spot scanner begins scanning in the discontinuousmicrobial growth area 18 of FIG. 1A. When the scanner is scanning in thecontinuous microbial colony area 26 of FIG. 1A, the total of the counter50 stays at a low number such as one or two. When the intercept counter50 repeatedly totals a number of intercept counts greater than two, itis an indication that the discontinuous area of microbial colony growthis being detected. While the number of the total count of the interceptcounter 50 which is chosen to indicate the interception of discontinuouscolonies for any one line of intersection is a matter of choice, asuitably programmed microprocessor can monitor the intercept counteroutput to signal the presence of discontinuous colony growth inaccordance with the foregoing analysis. The output of the AND gate 48 isapplied to an intercept length computation block 60 which totals thenumber of pulses outputted by the AND gate 48 during each revolution ofthe spot of the spot scanner 40. As described supra, the total number ofoutput pulses from AND gate 48 per scan revolution which are totaled bythe intercept length computation block 60 is a function of the densityof the microbial colonies along the line of intersection. The output ofthe intercept length computation block 60 ICi is applied on line 62 tothe growth ratio computation block 64 which computes the growth ratioGRi for each revolution of the spot scanner in accordance with therelationship GRi=(ΔR×ICi)/(Wo×N) wherein Wo (reference width) is themeasured width of the visible microbial colonies on the control plate, Nis the number of clock pulses on the output line 44 of the scanner 40per revolution of the scanner, ICi is the output from the interceptlength computation block 60 and ΔR is the radial advance betweensuccessive revolutions of the spiral of the interaction plate 10. Themathematically rigorous definition of ##EQU1## is not readily calculablewith the system of FIG. 3 because the measurement of the length ofinterception Li is not directly measurable. The invention calculates##EQU2## from the parameters ΔR, N, and ICi; which are available in thesystem of FIG. 3 as inputs to the growth computation box 64. As has beenexplained, supra, the length of interception Li, which is equal to thequantity ICi produced as an output of the length of interception box 60,is a measure of both the number and size of bacterial colonies.Determination of colony size and number is an important measure of theeffectiveness of a growth interacting substance. The quantity GRi is anindication of the effectiveness of the potency of the growth interactionsubstance in affecting growth on the interaction plate 10 along the ithscan of the spot scanner. A GRi of 1 indicates no growth effect; a GRi>1indicates an enhancement of growth; a GRi<1 indicates an inhibitoryeffect and a GRi of near or at zero indicates the point of inhibition 20of FIG. 2.

The potency computation Pi is made by utilizing known information fromthe programmed gradient deposition of the growth interacting substancereferred to as supra. The average scan radius Ri of each revolution ofthe spot scanner during intersection of the spot with the track 14 ofvisible colonies N on the interaction plate is computed by the averagescan radius computation block 66 which solves the equationRi=(Ro-1/2Δr)-ixΔr wherein Ro is the initial scan radius of the spotscanner, i is the number of the revolution of the scanner with therebeing 500 revolutions per plate and is the output from the scanrevolution counter 52 on line 65 and Δr is the spiral advance betweenadjacent revolutions of the scanner. The deposition factor DFi for eachline of intersection i of the spot with the visible colonies 12 on thetrack 14 is determined by the deposition factor computation block 68.This determination can be made from use of information such as that inthe appendix A, provided by Spiral System Instruments, Inc., ofdeposition factor values as a function of position along the spiraltrack for the particular SPIRAL PLATER model being used. Alternately, itcan be computed from an equation applicable to the SPIRAL PLATER beingused. For example, for the Model C SPIRAL PLATER in the 40 microliterdeposition mode, the deposition factor DFi can be computed from theequation DFi=[KD/(RA+12)]×10 EXP(-n×RA) microliters per millimetersquared, where KD=0.5746, n=0.039, and RA is the distance from thedeposition starting radius to the radial position of the point ofinterest on the spiral track. The potency Pi is calculated in responseto the deposition factor computation DFi by the potency computationblock 70 which solves the equation Pi=(RP×DFi)/h×SR) wherein h is theheight of the culture medium in the interaction plate 10, RP is thereference potency used in depositing the growth interacting substance inmicrograms per ml and SR is a correction factor to account for less thancomplete diffusion or spreading between adjacent turns of the track 14when less than a continuous gradient exists between adjacent turns. TheSR correction factor is the fraction of the spiral track separation ΔRwhich is covered by the growth interacting substance.

The value of SR can be measured by making two plates. In the firstplate, the sample is deposited directly over the spiral pattern while,in the second plate, the growth interacting substance is deposited inbetween the tracks of the spiral pattern (by rotating the turntable 180°relative to the starting position of the first plate). If there issignificant displacement of a point of interest on the inhibition tail23 such as the point of inhibition B at radius R₁ in FIG. 2 for therespective plates, the ratio of the corresponding value of DF(i) willgive a quantitative value of SR.

The output computations performed by the output computation block 58involve data analysis of the potency and growth ratio computationsperformed by the growth ratio block 64 and the potency computation block70. As described supra, the point where the microbe colony growthchanges from continuous to discontinuous growth is detected bymonitoring the count of the intercept counter 50. The point ofinhibition may be detected by monitoring the output line 46 of thespiral spot scanner to detect when no new visible microbe colonies havebeen detected after a given number of revolutions of the spot scanner40. The number of revolutions which must be completed without detectinga new colony to signal an indication of the point of inhibition is amatter of choice. The actual implementation of the detection of thepoint of inhibition 20 may be accomplished by a suitably programmedmicroprocessor. The computation performed by the output computationblock 58 may also be either local growth measurements LGM for a singleline of intersection of the spot with the visible microbe colonies 12 ofthe spiral track 14 or a regional growth measurement RGM which is theaverage value of a number of contiguous local growth measurements. Thecomputation performed by the output computation block 58 may alsoinvolve the detection of points of limitation e.g. maximum or minimumand slopes of the type illustrated in FIGS. 4A, 4B, and 4C discussedinfra. The outputs of the output computations block are applied to aplotter 72 which produces the curves of FIGS. 4A-4C.

It is important to assure alignment of the center of the interactionplate and the center of the scan radius. One way of detecting if properalignment exists is to monitor the growth ratio GR versus potency Pcurve from the output of the computation block 58 for the presence of aperiodic component which is indicative of misalignment. For example,such a periodic component would manifest itself in the growth ratio GRversus potency P curve of FIG. 4A as a periodic component in the linesegment SCI. The detection of a periodic component can be visuallyobserved or signalled. The operator would then be aware that alignmentcorrection was required and would take appropriate steps to check theplacement of the interaction plate on the scanner. If this proceduredoes not eliminate the misalignment, a recalibration of the alignment ofthe SPIRAL PLATER and the scanner 40 should be performed.

FIG. 4A illustrates the growth curve which is typically obtained by useof the invention to analyze interaction plates for growth inhibitinginteracting substances such as biocides or antibiotics. The growth curvehas essentially three distinct regions. The area SCI represents thecontinuous growth region of the visible colonies of microbes. The areaSDI represents the discontinuous growth region of the visible coloniesof microbes. The point PT(I) represents the point of inhibition whichmay be chosen to represent a GR value of zero or approaching zero. Thepotency PCD(I) represents the transition between continuous anddiscontinuous microbial growth. The potency PD(I) represents anextrapolation of the linear discontinuous region of microbial colonygrowth to zero growth ratio. As the potency of the growth interactingsubstance continues to increase, the linear slope SDI gives away androlls off toward zero. The potency PC(I) represents a linearextrapolation of the SCI continuous growth region.

FIG. 4B illustrates the growth curve which is typically obtained by useof the invention to analyze interaction plates for growth enhancinginteracting substances such as vitamins. Like FIG. 4A, the growth curveis comprised of essentially three segments. The first segment SDErepresents the region of low density visible microbial colonies wherethere is a slight enhancement of microbial growth as represented by agrowth ratio larger than one. The next region SCE represents an areawhere there is a sharp linear increase in growth ratio as a function ofincreasing potency. In this region the microbial colony growth becomesmore dense. The potency PC(E) represents an extrapolated potency of theSCE region. Finally, as the potency increases past PCM(E) the growthratio approaches a maximum GRM.

FIG. 4C illustrates the growth curve which is typically obtained by useof the invention to analyze interaction plates for mutagenic growthinteracting substances. As is apparent from inspection of FIG. 4C, amutagen can have both growth enhancing and inhibiting characteristicswhich vary as a function of potency. The region SDE represents a regionof linear enhancement of growth by the mutagenic substance. The growthcurve changes slope at potency PCD(E) to a region SCE of more sharplyenhanced growth. The maximum growth ratio is represented by thereference GPM which occurs at a potency PC(IE). As the potency continuesto increase, the mutagenic substance becomes inhibitory in region SCI.The inhibition in region SCI falls off with increasing potency until thepotency PCD(I) is reached where the inhibitory effect becomes morepronounced in region SDI where the growth ratio falls off sharply untilit approaches zero at potency PT(I). Potency PD(I) is the potencyrepresented by the extrapolation of region SDI to a growth ratio ofzero. The growth ratio GRM(IE) represents an extrapolation of the SCEand SCI regions.

FIGS. 4A-4C represent a wide range of typical growth curves which may beobtained by use of the invention to analyze interaction plates. With theinvention the foregoing types of curves may be obtained directly fromplotting the information available at the output computations block 58of FIG. 3.

It should be understood that the schematic illustrated in FIG. 3 isintended to illustrate the functions to be performed by the invention.In the preferred form of the invention, the calculations performed bythe various computation devices of FIG. 3 would be made by a suitablyprogrammed microprocessor which would perform each requisite calculationtask serially in a single computation loop. The programming of amicroprocessor to perform the computation tasks of FIG. 3 does notconstitute part of the invention and is routinely accomplished by acomputer programmer.

It should be understood that while the preferred form of the inventionuses spiral patterns for the deposition of the growth interacting andmicrobe containing solutions, and for scanning the incubated plate otherdeposition and/or scanning patterns may be used without departing fromthe spirit and scope of the invention. Also, while reference is madeherein to a spot scanner, it should be understood that the invention maybe used in conjunction with multiple spots used to scan simultaneously.Accordingly, the term "spot", as used in the specification and in theclaims, covers either a single spot or multiple scot scanner.

The following example illustrates the use of the method of the firstembodiment of the invention.

EXAMPLE

A suspension of B. Subtilus VAR Niger spores in a concentration of aboutone million per ml was deposited at a uniform rate on a 10 cm petri dishcontaining 20 ml of Mueller Hinton agar with a Spiral Systems, Inc.SPIRAL PLATER Model C with the spacing between spirals set to be 3.1 mm.Then a Streptomycin solution of 400 mg/ML was deposited on top of thebacterial spiral pattern. The resultant plate was incubated forapproximately 20 hours which produced a partial spiral of visiblecolonies including both a contiguous growth section and a discontinuousgrowth section. The radius of the point of inhibition was located andmeasured at approximately 11 mm. The measured radius of 11 mm was usedto compute the average concentration of Streptomycin in the column ofagar below the point of inhibition of about 1.1 micrograms per ml byusing published data pertaining to the model of the SPIRAL PLATER used.This value was used to represent the minimum inhibitory concentration ofStreptomycin acting on the microbes in the tested concentration.

The foregoing example is illustrative of the method of the firstembodiment of the invention. The first embodiment of the invention isapplicable to determining the point of inhibition of other microbes incombination with other growth interacting substances. The firstembodiment may also be used to determine the potency of the growthinteracting substance for any point of interest within the visiblemicrobial colonies on an interaction plate. Thus, with reference to FIG.1A, the potency Pi of the growth interacting substance at the point ofinhibition 20 is determined by measuring the radius between the centerof the interaction plate and point 20 and correlating the measuredradius with the published figures referred to supra. Specifically, asset forth, supra, the equation Pi=(RP×DFi)/(h×SR) is solved for eachpoint of interest. The deposition factor DFi is obtained from Appendix"A" of the aforementioned paper for the radial distance RA between thestarting radius of the spiral track to the point of interest. The factorSR is obtained as set forth above and h is the thickness of the culturemedium in the interaction plate. Similarly, the potency at other pointsof interest of the growth interacting substance is determined bymeasuring the radius between these points and performing theaforementioned correlation of the point of interest with the publisheddata.

What we claim is:
 1. A method for determining the effect of a growthinteracting substance on microbial growth by utilizing an interactionplate which contains visible microbial colonies in a track produced bythe programmed deposition of a microbe containing solution on a track onthe plate and the programmed deposition of a track of a growthinteracting substance on the interaction plate in contact with themicrobes and incubating the plate to produce visible colonies ofmicrobes comprising:(a) scanning the interaction plate with a scannerwhich scans a spot in a series of adjacent lines to detect a length ofinterception Li of the spot with the visible colonies of microbes toproduce a measurement of the number and size of visible colonies along aline of intersection of the spot with the track; (b) calculating thewidth of the track of visible colonies of microbes along the line ofintersection of the spot with the track from the detected length ofinterception; and (c) calculating the growth ratio GRi for eachintersection of the spot with the track of visible colonies of microbesby calculating a ratio of the width of the track for the line ofintersection of the spot with the interaction plate and a referencewidth.
 2. A method in accordance with claim 1 further comprising:(a)calculating the growth interacting substance potency Pi along the lineof intersection of the spot with the track of visible colonies ofmicrobes; and (b) plotting the potency Pi versus growth ratio GRi forsuccessive scans of the spot scanner of the interaction plate to producea growth curve.
 3. A method in accordance with claim 1 wherein:(a) thetrack is a spiral with a radial advance ΔR per revolution of the spiral;(b) the interaction plate has a gradient of deposition of the growthinteracting substance which decreases in volume per unit length as theradius of the spiral track increases; and (c) the microbe containingsolution concentration is constant per unit length of the track.
 4. Amethod in accordance with claim 3 wherein the calculation of the widthWi of the track of visible microbe colonies for each scan of theinteraction plate is made in accordance with the equation Wi=ΔR×ICi/Nwhere ΔR is the radial advance between successive revolutions of thespiral of the interaction plate, N is the number of clock pulsesproduced per revolution of the scanner and ICi is equal to the length ofinterception of the spot along the ith revolution of the scanner.
 5. Amethod in accordance with claim 4 wherein the spot scanner scans incircular patterns which change in radius by the uniform distance Δrbetween successive revolutions of the spot.
 6. A method in accordancewith claim 4 wherein the spot scanner scans in a spiral pattern ofuniformly changing distance Δr per revolution of the spot.
 7. A methodin accordance with claim 3 further comprising:(a) detecting the point onthe interaction plate where visible microbial growth is inhibited; (b)calculating the growth interacting substance potency Pi for the point onthe interaction plate where visible microbial growth is inhibited; and(c) calculating the growth ratio GRi where visible microbial growth isinhibited.
 8. A method in accordance with claim 3 further comprising:(a)detecting the point on the interaction plate where the visible microbialgrowth changes from continuous colonies to discontinuous colonies; (b)calculating the growth interacting substance potency Pi for the pointwhere the visible microbial growth changes from continuous todiscontinuous; and(c) calculating the growth ratio GRi for the pointwhere the visible microbial growth changes from continuous todiscontinuous.
 9. A method in accordance with claim 3 further comprisinganalyzing the growth curve to quantitate selected growth curveparameters.
 10. A method in accordance with claim 1 wherein the growthinteracting substance is a biocide.
 11. A method in accordance withclaim 1 wherein the growth interacting substance is an antibiotic.
 12. Amethod in accordance with claim 1 wherein the growth interactingsubstance is a mutagen.
 13. An apparatus for measuring the effect of agrowth interacting substance on microbial growth by utilizing aninteraction plate which contains visible colonies of microbes in a trackproduced by the programmed deposition of a microbe containing solutionin a track on the plate and the programmed deposition of a track of thegrowth interacting substance on the interaction plate in contact withthe microbes and incubating the plate to produce visible colonies ofmicrobes comprising:(a) scanning means for scanning a spot in a seriesof adjacent lines for detecting the visible colonies of microbes along aline of intersection of the spot with the track of visible colonies ofmicrobes; (b) means responsive to the scanning means for measuring alength of interception Li of the spot with the visible colonies ofmicrobes to produce a measurement of the number and size of visiblecolonies along the line of intersection of the track of visiblecolonies; (c) means responsive to the means for measuring a length ofinterception for calculating the track width Wi of visible colonies ofmicrobes for each line of intersection of the spot with the visiblecolonies; and (d) means for calculating the growth ratio GRi for eachline of intersection of the spot with the track of visible colonies ofmicrobes by calculating a ratio of track width for the line ofintersection of the spot with the track of visible colonies and areference width.
 14. An apparatus in accordance with the claim 13further comprising:(a) means for calculating the growth interactionsubstance potency Pi at each line of intersection of the spot with thetrack of visible colonies of microbes; and (b) means for plotting thepotency Pi versus growth ratio GRi for successive lines of intersectionof the spot with visible colonies.
 15. An apparatus in accordance withclaim 13 further comprising:(a) an interaction plate; (b) each track isa spiral with a radial advance ΔR per revolution of the spiral; (c) theinteraction plate has a gradient of deposition of the growth interactingsubstance which decreases in volume per unit length as the radius of thespiral track increases; and (d) the microbe containing solutiondeposited per unit length of the track is constant.
 16. An apparatus inaccordance with claim 15 wherein the means for calculating average trackwidth Wi solves the equation Wi=ΔR×ICi/N where ΔR is the radial advancebetween successive revolutions of the spiral of the interaction plate, Nis the number of clock pulses produced per revolution of the scanner andICi is equal to the length of interception of the spot along the ithrevolution of the scanner.
 17. An apparatus in accordance with claim 16wherein the scanning means is a circular spot scanner which scans incircular patterns which change in radius by the uniform distance Δrbetween successive lines of the spot.
 18. An apparatus in accordancewith claim 16 wherein the scanning means is a spiral spot scanner whichscans a spot in a uniformly changing radius of distance Δr perrevolution of the spot.
 19. An apparatus in accordance with claim 15further comprising:(a) means for detecting the point on the interactionplate where visible microbial growth is inhibited; (b) means forcalculating the growth interacting substance potency Pi for the point onthe interaction plate where visible microbial growth is inhibited; and(c) said means for calculating calculates the growth ratio GRi for thepoint where the growth of the visible colonies of microbes is inhibited.20. An apparatus in accordance with claim 15 further comprising:(a)means for detecting the point on the interaction plate where the visiblecolonies of microbes change from continuous colonies to discontinuouscolonies; (b) means for calculating the growth interacting substancepotency Pi for the point where the visible colonies of microbes changefrom continuous to discontinuous; and (c) said means for calculatingcalculates the growth ratio GRi for the point where the visible coloniesof microbes change from continuous to discontinuous.
 21. An apparatus inaccordance with claim 16 or 17 wherein the means for measuring thelength of interception comprises:(a) an AND gate having two inputs andan output, the first input being an output signal from the scanningmeans of the visible colonies of microbes, and the second input signalbeing clock pulses from scanning means; (b) means responsive to clockpulses from the scanning means for signaling the end of scanning of eachline; and (c) means responsive to the output of the means for signalingthe end of scanning of each line and the output of the AND gate forsumming the number of clock pulses during which the spot of the scanningmeans is intersecting visible colonies of microbes during each line ofscanning, the means for signaling having an output coupled to the meansfor calculating the growth ratio GRi.
 22. An apparatus in accordancewith claim 21 further comprising:(a) means for counting the number ofthe line scan of the scanning means; (b) means for calculating thegrowth ratio GRi having an input coupled to the clock pulses from thescanning means; (c) means for calculating the average scan radius Ri foreach line of intersection of the spot with the track of visible coloniesof microbes, the means for calculating the average scan radius Ri havingan input coupled to an output of the means for signaling the end ofscanning of each line, an input of the initial scan radius Ro and aninput of Δr and solving the equation Ri=(Ro-1/2Δr)-iΔr where i is thenumber of the line scan of the scanning means and is an output from themeans for counting the number of the line scan of the scanning means andΔr is the change in radius of the scanner per line scan of the scanningmeans; (d) means for calculating the deposition factor DFi for each lineof intersection of the spot with the track of visible colonies ofmicrobes of the interaction plate having inputs of the average scanradius Ri, KD, and n where KD and n are constants of an apparatus usedfor depositing the spiral track, RA is the distance from the depositionstarting radius to the radial position of the point of interest on thespiral track and DFi=×10 EXP (-n×RA); and (e) means for calculating thegrowth interacting substance potency Pi which is responsive to the meansfor calculating the deposition fact DFi, the means for calculating thegrowth potency Pi having inputs RP, h, and SR wherein RP is thereference potency of the growth interacting substance, h is thethickness of the culture medium on the interaction plate and the controlplate, if used, and SR is a correction factor to account for incompletediffusion between adjacent tracks.